Glioblastoma (GBM) is the most common primary brain tumor and also the highest grade (WHO grade IV). Progression to a GBM represents an abrupt turning point, with rapid progression to death following the transition (mean, 50 weeks). Two pathologic features that distinguish GBM from lower grade tumors and are mechanistically instrumental are necrosis, typically with surrounding cellular pseudopalisades, and microvascular hyperplasia. Pseudopalisades are hypoxic and secrete pro-angiogenic factors that promote microvascular hyperplasia, an exuberant form of angiogenesis that supports the rapid tumor expansion. Mechanisms underlying pseudopalisades, hypoxia, and necrosis in GBM have not been defined. We hypothesize that vaso-occlusion and intravascular thrombosis give rise to pseudopalisades and the ensuing hypoxia-induced angiogenic cascade, accounting for the abrupt onset of rapidly progressive disease. This proposal follows our preliminary data, which indicates that pseudopalisades are neoplastic cells migrating away from central hypoxia created in part by microscopic thrombotic vascular occulsion. The initiating vascular insult that precedes intravascular thrombosis and pseudopalisading necrosis has not been determined. Ang-2, a Tie-2 receptor antagonsist that mediates endothelial apoptosis and vascular regression in the absence of VEGF, is expressed by endothelial cells of high grade gliomas and is a prime candidate for initiating vascular pathology. Also occurring during the transition to GBM are PTEN mutations and increased tumor cellularity, especially around blood vessels. We will examine whether neoplastic PTEN loss leads to the secretion of a protein capable of triggering endothelial apoptosis through Ang-2 mediated mechanisms. Candidate secreted or cell contact proteins will be identified by advanced protein separation methods and mass spectrometry. We also hypothesize that intravascular thrombosis is relevant to tumor necrosis and glioma biology and will examine whether PTEN loss and/or hypoxia, promote thrombosis through increased expression of the pro-thrombotic factors PAR-1 and tissue factor. The rationale and experiments in this proposal are novel and innovative, since the etiologies of hypoxia, pseudopalisades, and necrosis are unknown, vascular mechanisms have not been proposed, and intravascular thrombosis, while a frequent finding, has not been recognized as a potential driving force in tumor progression. Our emerging model represents a paradigm shift in the understanding of GBM and should lead directly to more effective therapies.